Expression vector: pFl (MultiBAC expression system) with Strep-Strep-Sumo-TEV-hAgo2.
The pFl vector also expresses YFP under the p10 promoter to monitor expression level. The use of the MultiBac expression system in which proteases are disrupted could be particularly important in this case.Note: Low YFP expression (<90-95%) will result in very low yields and no RNA-free fraction!
SF9/Hi5 were infected for 72 hours.

Expression yields: Yields for hAgo2 bound to endogenous SF9/Hi5 RNA are ~ 1-2mg/L of cells (after strep purification). RNA free hAgo2 is usually no more than 10-15% of the total hAgo2 expression. In order to obtain significant amounts of RNA-free hAgo2, it is recommended to express >5L of SF9/Hi5 cells. The cation exchange chromatogram in Elkayam et. al (Cell 2012) is from a purification preparation of 8L of SF9 cells.

Purification:
1. SF9/Hi5 cells are re-suspended in the following buffer: KCl 100mM, Tris 50mM, DTT 5mM, and PI cocktail (tablets from Roche, EDTA free or home made cocktail) flash freeze in liquid N2 and store in -80°C.

Thaw the frozen cells inside water beaker at RT.
Add KCl to a final concentration of 400mM.
Sonicate for 1 min with 1 sec on 5 sec off at 70% amplitude in an ice water bath.
Add PEI to a final concentration of 0.2% (V/V), mix by inverting a few times, do not vortex!!!
Immediately spin at 35K for 45min using ultracentrifuge (rotor Ti 45).

Elute using a linear gradient of Tris 50mM pH 8.0, KCl 1000 mM, 5 mM DTT over 30 CV (this step is crucial to determine what step gradient to use next time, the KCL concentration can change slightly between different column between different cation exchangers.